The objective of the proposed research is to investigate the cellular and molecular mechanisms of hemoglobin switching using established lines of human hemoglobin synthesizing cells. A new erythroleukemia line (the HEL cells) which we have established will be mainly used. This line produces fetal and embryonic globin chains. Our aims are a) to characterize the HEL line in orders to define the potentialities of the cells. This will be pursued with studies of a variety of immunochemical, cytochemical and biochemical markers specific for the erythroid, myelomonocytic, lymphoid and megakaryocytic lineages or markers expressed in more than one lineage, b) to clone the HEL cells in order to test for clonal heterogeneity in globin expression or expression of lineage specific characteristics or differential responses to inducers, c) to produce homospecific hybrids of HEL or K562 cells with erythroid cells of persons with abnormal hemoglobins in order to test for presence of trans-acting regulators of beta and gamma synthesis and to study the relevance of these regulators to Hb switching d) to examine whether there is spontaneous expression of beta globin in HEL or K562 cells and to attempt to modulate globin expression in these cells using culture conditions which inhibit DNA methylation, e) to study expression of the globin program in the chromosomally marked progeny of cryopreserved cells which have been obtained from the patient in two stages of his disease, f) to attempt to establish hemoglobin synthesizing lines using cells from leukemia patients in post-transplantation relapse. The studies on hemoglobin expression of the HEL cells and the experimental manipulations we plan to do will shed light on the question of regulation of hemoglobin switching and may produce clones which will allow testing of the molecular events involved. The HEL line, characterized as we propose to do, will provide a new valuable tool for studies of hemopoietic cell differentiation.